Von Recklinghausen neurofibromatosis (NF1), often referred to as the "elephant man disease,".sup.1 is one of the most common autosomal dominant human disorders, affecting about 1 in 3,000 of the general population. The disease primarily involves neural crest-derived tissue and is characterized by cafe-au-lait spots, neurofibromas increasing in size and number with age, learning disabilities and mental retardation, seizures, and an increased risk of malignancy. The expression of the disease is extremely variable in its symptoms and severity, and the spontaneous mutation rate is remarkably high, with about 30 to 50% of all cases representing new mutations. Clinical diagnosis of NF1 has been relatively difficult early in life, due to the variability of the symptoms and their delayed appearance. FNT .sup.1 An NIH panel, however, recently concluded that "Elephant Man" J. Merrick did not actually suffer from neurofibromatosis, but from an extremely rare disease known as the Proteus syndrome.
Direct cloning of the NF1 gene has not been possible due to the lack of a consistent abnormality in NF1 tissue which would provide sufficient information about the gene product. The remaining alternative has been positional cloning of the gene, utilizing its chromosomal map position rather than its functional properties. Using this approach, genetic linkage analysis led to the assignment of the NF1 gene to the proximal long arm of chromosome 17. Subsequent collaborative multipoint mapping efforts narrowed its genetic location to about 3 centiMorgans of 17q11.2. A combination of somatic cell hybrid techniques, linking clones and pulsed field gel electrophoresis (PFGE) applied to two unrelated NF1 patients having balanced translocations t(1;17) and t(17;22), with breakpoints approximately 60 kb apart on chromosome 17, further narrowed the location of the gene to a few hundred kilobases of chromosome band 17q11.2. See Collins, F. S. et al., Trends in Genetics 5:217-221 (1989).
The first NF1 candidate gene was identified in mice as a site of retroviral integration in murine leukemia. See Buchberg, A. M. et al., Oncogene Research 2:149 (1988). It has now been found, however, that the human homolog EVI2A, previously named EVI2, which maps between the NF1 breakpoints, is not interrupted by the aforementioned NF1 translocations, and no abnormalities in this gene have been identified as the cause of NF1. Similarly, EVI2B, previously named NF1-c2, a gene newly identified in the course of this invention by chromosomal walking and jumping, mapped between the NF1 breakpoints, was not interrupted by the NF1 translocations and exhibited no abnormalities in NF1 patients. It thus became clear that the NF1 gene had not yet been identified.
Recently, a gene was identified by positional cloning showing mutations in individuals affected with NF1. Cawthon, R. et al., Cell 62:193-201 (1990); Viskochil, D. et al., Cell 62:187-192 (1990); Wallace, M. R. et al., Science 249:181-186 (1990). Further cloning and partial sequence analysis demonstrated that the gene product contains a domain showing approximately 30% similarity to the catalytic domains of yeast IRA1 and IRA2 proteins and the mammalian GTPase activating protein (GAP). Buchberg, A. et al., Nature 347:291-294 (1990); Xu, G. et al., Cell 62:599-605 (1990). GAP is a cytosolic protein that catalyzes the conversion of active GTP-bound ras p21 to the inactive GDP-bound form. Trahey M. et al., Science 238:542-545 (1987). It was subsequently shown that the GAP related domain of the NF1 gene product can also interact with human and yeast RAS p21 to down-regulate its activity. Ballester, R. et al., Cell 63:851-859 (1990); Martin, G. A. et al., Cell 63:343-349 (1990); Xu, G. et al., Cell 63:835-841 (1990). Our previous reports of cDNA cloning of NF1 contained in parent application U.S. Ser. No. 547,090 were based on partial fragments of the transcript which is approximately 13 kb by Northern blotting. Wallace, M. R. et al., Science 249:181-186 (1990). The entire coding region of the NF1 gene has now been cloned and sequenced, the gene product identified and antibodies raised thereto, as described and claimed herein.